Ultraspiracle of the stingless bees Melipona scutellaris and Scaptotrigona depilis: cDNA sequence and expression profiles during pupal development

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and Scaptotrigona depilis: cDNA sequence and

expression profiles during pupal development

Aline Carolina Aleixo Silva Teles, Tathyana Rachel Palo Mello, Angel Roberto

Barchuk, Zilá Luz Paulino Simões

To cite this version:

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Apidologie 38 (2007) 462–471 Available online at: c

INRA/DIB-AGIB/ EDP Sciences, 2007 www.apidologie.org DOI: 10.1051/apido:2007035

Original article

Ultraspiracle of the stingless bees Melipona scutellaris and

Scaptotrigona depilis: cDNA sequence and expression

profiles during pupal development*

Aline Carolina Aleixo Silva T

eles

b

_{, Tathyana Rachel Palo M}

ello

b

_,

Angel Roberto B

archuk

a,b

, Zilá Luz Paulino S

im˜oes

b

a_{Depto. Ciências Biológicas, Universidade Federal de Alfenas. Rua Gabriel Monteiro Silva 714}

-Cep 37130-000 Campus Unifal, Alfenas, Minas Gerais, Brazil

b_{Depto. de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,}

Universidade de São Paulo, Brazil

Received 15 January 2007 – Revised 11 June 2007 – Accepted 19 June 2007

Abstract – Reproduction and development in insects and caste diﬀerentiation in bees are mainly

gov-erned by ecdysteroids and juvenile hormone. We characterized the ultraspiracle (nuclear hormone recep-tor) cDNA of the highly social bees Melipona scutellaris and Scaptotrigona depilis. The predicted Usp proteins (with 427aa) show a greater sequence similarity to its orthologs from the “ApLoTe” group than to the dipteran-lepidopteran group, suggesting that Usp proteins included in the bee group might share func-tional characteristics. The expression profiles in fat bodies (FB) and brains turned out not to be very similar between the species. In M. scutellaris FB and brain and in S. depilis brain usp expression anti-parallels pupal ecdysteroid titers, thus suggesting a repression of usp expression by this hormone.

ultraspiracle/ juvenile hormone / ecdysone / vitellogenin / fat body / brain / Apidae

1. INTRODUCTION

Insect reproduction and development are mainly governed by the lipophilic hormones ecdysteroids and juvenile hormone (JH). In some highly eusocial bees, JH is also respon-sible for the developmental processes leading to caste diﬀerentiation (Velthuis and Velthuis-Kluppell, 1975; Barchuk et al., 2007) and for major behavioral switches occurring in adult workers (Robinson and Vargo, 1997). Ecdys-teroids bind nuclear receptor proteins (belong-ing to the nuclear hormone receptors super-family) inside the cell which, in turn, results in the activation or the repression of target genes

Corresponding author: A.R. Barchuk, barchuk@unifal-mg.edu.br,

arbarchuk@yahoo.com, barchuk@rge.fmrp.usp.br * Manuscript editor: Stefan Fuchs

(Koelle et al., 1991). Ultraspiracle (Usp), one of the proteins that constitutes the ecdysone receptor complex together with the ecdysone receptor protein (EcR; Oro et al., 1990; Yao et al., 1993), has ligand-dependent activity in Drosophila melanogaster (Jones et al., 2006) and has been suggested to have it in Apis mel-lifera (Barchuk et al., 2004). By heterodimer-izing with EcR and HR38 (an orphan nuclear receptor; Sutherland et al., 1995) and by ho-modimerizing and binding JH (Jones et al., 2006), Usp becomes a pleiotropic regulator of insect development, mediating the action of both main hormones in insects, ecdysteroids and JH. For a review on nuclear hormone re-ceptors in the honeybee A. mellifera, see the recent work of Velarde et al. (2006).

Melipona scutellaris Latreille and Scap-totrigona depilis Moure are stingless honey-bees which, like stinging honeyhoney-bees, possesses Article published by EDP Sciences and available at http://www.apidologie.org

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high levels of social organization and whose caste diﬀerentiation processes are governed by JH and ecdysteroids (see Engels and Imperatriz-Fonseca, 1990; Hartfelder et al., 2006). One of the main phenotypic manifesta-tions of caste diﬀerentiation in bees that under-lies the reproductive division of labor among females is the production of vitellogenin (vg), the main protein of insect eggs (see Engels et al., 1990). The expression of the vg gene has been extensively studied in A. mellifera (Engels et al., 1990; Barchuk et al., 2002; Piulachs et al., 2003; Guidugli et al., 2005a, b). The onset of vg expression during pupal development in this species occurs in the con-text of an increasing JH titer and decreasing levels of ecdysteroids (Barchuk et al., 2002; Piulachs et al., 2003). Thus, Usp protein may be mediating the regulation of vg expression via its participation in the ecdysone receptor complex or acting as JH receptor. In stingless bees, the expression of the vg gene associated with workers’ egg-laying capacity has been studied in S. postica depilis (S. depilis) and M. scutellaris (Dallacqua et al., 2006; Hartfelder et al., 2006; Hartfelden, personal communica-tion). The authors showed that in both species (and in Frieseomellita varia) vg is expressed during the entire pupal stage and adult life. Ju-venile hormone and ecdysteroid titers during post-embryonic development have also been determined for these bees, specifically S. de-pilis and M. quadrifasciata (Hartfelder and Rembold, 1991; Pinto et al., 2002).

Another phenotypic manifestation of caste differentiation governed by hormones and their nuclear receptors in bees is related to the degree of brain development. The differential nervous system development in workers favors learning and memory-related skills that bees use for navigation, foraging, kin recognition and other activities. In insects, the main ner-vous system organ in charge of these func-tions is the mushroom body (MB), an inte-grative part of the brain (Fahrbach, 2006). The differential growth in MB between queens and workers includes differential proliferation and fasciculation. The high titers of ecdys-teroids during larval development in A. mel-lifera queens (Hartfelder and Engels, 1998) likely impair a pronounced MB development,

since the mitotic activity of MB cells has been shown to be inhibited by 20E (Ganeshina et al., 2000; Malun et al., 2003).

Multiple alignment analyses have shown that the ligand-binding domain of A. mellifera Usp is more similar to that of vertebrates RXR (which binds 9-cis retinoic acid; Mangelsdorf et al., 1992) than to those of other insects (diptera and lepidoptera), suggesting the ex-istence of a not yet identified ligand for this nuclear receptor in honeybees (Barchuk et al., 2004). Thus, the existence of a ligand that may induce the activity of a transcription fac-tor can be suggested by phylogenetic studies (see Escriva et al., 2004) and may eventually be demonstrated by biochemical and physio-logical studies.

Here we characterized the ultraspiracle cDNA of M. scutellaris and S. depilis, which are members of the Apidae family and bees be-ing used in comparative and evolutionary stud-ies of insect polyphenism (Hartfelder, 1987; Hartfelder and Rembold, 1991; Hartfelder and Emlen, 2005; Dallacqua et al., 2006; Vieira et al., 2006; Hartfelder et al., 2006; Cruz-Landim et al., 2006). Our aim was to obtain information about its amino acid sequence, which would allow us to infer infor-mation about the eventual existence of ligand-dependent activity of this nuclear hormone receptor in these bees, thus facilitating evolu-tionary studies. In addition, and with a view to shed light on the molecular mechanisms con-trolling vg expression during pupal stages and the brain development of these bees, we deter-mined the expression profile of usp gene in fat bodies and brains during worker pupal devel-opment.

2. MATERIALS AND METHODS 2.1. Bees

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464 A.C.A.S. Teles et al.

vg synthesis takes place) and brains were extracted in dissecting dishes on dry ice, suspended in TRI-zol reagent (Invitrogen) and frozen at –80◦C until RNA isolation.

2.2. Cloning and sequencing the

ultraspiracle cDNAs and

bioinformatics

Total RNA was extracted from fat bodies and brains using a TRIzol (Invitrogen) protocol or al-ternatively the GenElute Mammalian Total RNA kit (Cat. RTN70, Sigma) and then incubated in the presence of DNAse I (Promega) for 30 min at 37◦C to eliminate contaminating DNA.

The cloning of Usp cDNAs was performed on PCR amplified nucleic acids obtained using primers based on the sequence of Usp cDNA from A. mellifera workers (Barchuk et al., 2004). Database searches were carried out at the National Center for Biotechnology Information using the BLAST server with orthologs aligned by ClustalW. For the molecular phylogeny analysis of Usp, the ClustalW results were converted into Mega 3.1 for-mat (Kumar et al., 2001). Tree construction was per-formed using the Maximum Parsimony procedure and Schistosoma mansoni Usp as out-group.

2.3. Semi-quantitative assay by RT-PCR

First-strand cDNA was synthesized as in Nasci-mento et al. (2004). Forward N_Usp-1 (5’-TCAAGAGGACCGTACGCAAG-3’) and reverse N_Usp-2 (5’-AACAGGTACTCCGGG CAGTT-3’) primers were utilized in PCR reactions for usp ex-pression quantification. The thermal cycling pro-gram was adjusted to the specific primer require-ments. For normalization we performed RT-PCR on β-actin gene using Act forward (5’ TGC-CAACACTGTCCTTTCTG 3’) and Act reverse (5’ AGAATTGACCCACCAATCCA 3’) primers based on A. melliferaβ-actin gene (GenBank accession number AB023025). The amplification products, an ∼ 800 bp sequence for Usp and ∼ 200 bp sequence forβ-actin, were run in 1% agarose gels with ethid-ium bromide. Band intensities were estimated using Tnimage Image Analysis 3.3.7a, and usp transcript levels were normalized against the respective lev-els ofβ-actin, whose expression varies only slightly during development.

3. RESULTS

3.1. Ultraspiracle cDNAs and their predicted translation products

The sequenced Usp cDNAs from M. scutel-laris and S. depilis workers have 1284 and 1665 bp, respectively, including an open reading frame coding for 427 amino acids. Both sequences have been deposited in GenBank under the accession numbers AY840093 (M. scutellaris) and DQ190542 (S. depilis). The predicted translation prod-ucts have an estimated pI value of 8.80 and 8.87 (respectively) and a molecular weight of approximately 48 kDa (pI/Mw tool, http://www.expasy.org/tools/pi_tools.html).

The sequenced Usp cDNAs from M. scutel-laris and S. depilis encode very similar polypeptides. The high level of similarity with those of other animal species (see Figs. 1, 2) and the modular organization of the trans-lated proteins indicate that the cDNAs code for a member of the nuclear hormone re-ceptor superfamily, specifically for the ultra-spiracle protein, an ortholog of the vertebrate RXR (Henrich and Brown, 1995; Riddiford et al., 2000; King-Jones and Thummel, 2005). The stingless bees Usp, thus, include the 4 main domains of this kind of transcription fac-tor (Fig. 1): the N-terminal A/B, C domain (DBD, DNA-binding domain), D (hinge do-main), and the carboxiterminal E/F domains (LBD, ligand-binding domain).

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ATGATGAAGAAGGAGAAGCCGATGATGTCGGTGACCGCTATCATCCAAGGGACCCAGGCT - 60 M M K K E K P M M S V T A I I Q G T Q A CAGCATTGGTCCCGTGGGAACACTTGGCTGAGTTTGGACAACAGTAACATGTCCATGTCC - 120 Q H W S R G N T W L S L D N S N M S M S TCGGTGGGCCCGCAAAGTCCGCTGGACATGAAGCCTGACACAGCTAGCCTCATCAATCCA - 180 S V G P Q S P L D M K P D T A S L I N P GGAAACTTCAGTCCTTCCGGTCCCAATAGTCCTGGATCCTTCACCGCTGGTTGTCACAGC - 240 G N F S P S G P N S P G S F T A G C H S AACCTTCTGAGTACGTCGCCGAGTGGACAGAACAAAGCGGTCGCGCCCTATCCGCCGAAC - 300 N L L S T S P S G Q N K A V A P Y P P N CACCCTCTATCTGGCAGCAAGCACCTCTGCTCGATCTGCGGTGACCGTGCCAGTGGCAAG - 360 H P L S G S K H L C S I C G D R A S G K CATTACGGTGTCTATAGCTGCGAGGGTTGCAAGGGTTTCTTCAAGAGGACCGTACGCAAG - 420 H Y G V Y S C E G C K G F F K R T V R K GACCTGTCGTACGCGTGCCGCGAGGAGAAGTCGTGCATCATCGACAAACGACAGAGAAAC - 480 D L S Y A C R E E K S C I I D K R Q R N CGATGCCAGTATTGCAGATACCAGAAATGCCTGGCGATGGGCATGAAGAGAGAAGCGGTC - 540 R C Q Y C R Y Q K C L A M G M K R E A V CATGAAGAACGCCAGCGTACCAAGGAGAGGGATCAGAGCGAGGTGGAGAGCACCAGTAGC - 600 H E E R Q R T K E R D Q S E V E S T S S TTGCACTCGGACATGCCGATCGAGCGCATCCTCGAAGCCGAGAAACGAGTCGAATGCAAA - 660 L H S D M P I E R I L E A E K R V E C K ATGGAACAGCAAGGGAATTACGAGAATGCAGTGTCGCACATTTGCAACGCCACGAACAAA - 720 M E Q Q G N Y E N A V S H I C N A T N K CAGCTGTTCCAGCTGGTAGCATGGGCGAAACACATCCCGCATTTTACGTCGTTGCCACTG - 780 Q L F Q L V A W A K H I P H F T S L P L GAGGATCAGGTACTTCTGCTCAGGGCCGGTTGGAACGAGTTGCTGATAGCCTCCTTTTCC - 840 E D Q V L L L R A G W N E L L I A S F S CACCGTTCCATCGACGTGAAGGACGGTATCGTGCTGGCCACGGGGATCACCGTGCACCGG - 900 H R S I D V K D G I V L A T G I T V H R AACTCGGCGCAGCAGGCTGGCGTCGGCACGATATTCGACCGAGTGCTTTCCGAGCTTGTG - 960 N S A Q Q A G V G T I F D R V L S E L V TCGAAAATGCGCGAAATGAAGATGGACAGGACGGAGCTTGGCTGTTTGAGATCTATAATA - 1020 S K M R E M K M D R T E L G C L R S I I CTCTTCAATCCCGAGGTCCGAGGACTGAAATCCATCCAGGAAGTGACCCTGCTCCGCGAG - 1080 L F N P E V R G L K S I Q E V T L L R E AAGATCTACGCAGCTCTGGAGGGTTATTGCCGCGTAGCCTGGCCGGACGACGCTGGAAGA - 1140 K I Y A A L E G Y C R V A W P D D A G R TTCGCGAAGTTGTTGTTACGCTTGCCGGCCATCCGATCGATAGGACTAAAGTGCCTCGAG - 1200 F A K L L L R L P A I R S I G L K C L E TACCTGTTCTTTTTCAAAATGATCGGGGACGTGCCGATCGACGACTTCCTCGTCGAGATG - 1260 Y L F F F K M I G D V P I D D F L V E M CTAGAGTCACGATCGGACCCTTAG - 1284 L E S R S D P *

Figure 1. Nucleotide and conceptual amino acid sequences of usp cDNA from the stingless bees M.

scutel-laris and S. depilis. The predicted polypeptide shown by a one-letter code below the respective nucleotide

sequence corresponds to M. scutellaris usp. The diﬀerences respect to the sequences of S. depilis are as follows: the nucleotide with an “A” in position 533 is a “G” resulting in a glycine (G) in place of the gluta-mate (E); and the “T” in position 543 is a “G”, resulting in a glutamine (Q) in place of the histidine (H) (the respective codons are underlined). Amino acids belonging to the conserved DBD (C domain) are shaded in light grey and those belonging to E/F domain are shaded in dark grey. Conserved amino acid sequences in C, D and E/F domains are bold and italicized.

glutamic acid (E); and in position 543 there is a “g”, resulting in the polar amino acid glu-tamine (Q) in place of the positively charged histidine (H).

The molecular phylogeny analyses of DBDs resulted in phylogenetic relationships

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Figure 2. Dendrogram based on entire proteins

analysis using Maximum Parsimony procedure, illustrating the evolutionary relationships among USPs/RXRs. A. mellifera = AY273778; M.

scutel-laris = AY840093; S. depilis = DQ190542; L. migratoria = AF136372; T. molitor =

AJ251542; C. pugilator = AF032983; D.

re-rio= AAC59720; M. musculus = XM_123763; H. sapiens= NM_002957; C. tentans = AF045891; D. melanogaster= X53417; L. cuprina = AY007213; C. suppressalis = AF016368; S. frugiperda =

U06073; S. mansoni= AF158102.

Tenebrio and Celuca and the RXR of verte-brates (ApLoTe group, Barchuk et al., 2004) clearly separated from the rest of insects Usp, like dipterans and lepidopterans (Fig. 2).

3.2. Expression profile of M. scutellaris and S. depilis usp in fat bodies and brains

We determined the expression profile of usp in fat bodies and brains during pupal develop-ment (Fig. 3). There was found little similarity between the species, except for the fact that the highest levels of usp expression in both organs and species are concentrated in the earliest stage of pupal development (Pw; see Tab. I), generally diminishing in the last stages. In-terestingly, in M. scutellaris FB and brain and in S. depilis brain usp expression anti-parallels pupal ecdysteroid titers found in M. quadrifasciata and S. depilis hemolymph (see Hartfelder and Rembold, 1991; Pinto et al., 2002; Hartfelder et al., 2006). A) 0 20 40 60 80 100 Pw Pp Pbl Pbm Pbd Pupal stages us p FB Brain B) 0 20 40 60 80 100 Pw Pp Pb Pbd Pupal stages us p FB Brain

Figure 3. Usp expression profiles in fat bodies (FB)

and brains during pupal development of M.

scutel-laris (A) and S. depilis (B). The ordinate represents

densitometric units and each value is an average of three separate experiments. SEMs are indicated by vertical bars. See Table I for the complete name of developmental stages.

4. DISCUSSION

4.1. Ultraspiracle from highly eusocial bees might share functional characteristics

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Table I. External morphological characters used to stage developing pupae of M. scutellaris and S. depilis

workers (see Dallacqua et al., 2006).

Pupal stage Eye pigmentation Body pigmentation

(Larva)

Pw white unpigmented

Pp pink unpigmented

Pb brown unpigmented

Pbm dark brown pigmented appendages; light-pigmented thorax and abdomen Pbd dark brown dark-pigmented appendages; light-pigmented thorax and abdomen (Adult)

domain could be assessed by testing Usp be-havior using recombinant molecules for the hinge domain or even the original proteins in cell culture assays. Nevertheless, since the D domain does not seem to have critical tions, these diﬀerences might not confer func-tional divergences in Usp of these two bee species (Henrich and Brown, 1995; Riddiford et al., 2000).

The molecular phylogeny analyses of the entire proteins (Usp/RXR), shows bees Usp together with that of Locusta, Tenebrio and Celuca and the RXR of vertebrates (ApLoTe group, Barchuk et al., 2004) separated from the rest of insects Usp. These results suggest a major contribution of the LBD on this clus-tering and that the Usp proteins included in the bee group might share functional characteris-tics.

Since the vertebrate RXR (Usp ortholog) has ligand-binding capacity, binding the 9-cis retinoic acid (Mangelsdorf et al., 1992), bees Usp might also have ligand-binding ac-tivity, as suggested by the sequence simi-larity among Usp from the ApLoTe group (see Barchuk et al., 2004; Fig. 2). Moreover, more than one ligand, with diﬀerent aﬃni-ties can exist within bees and other insect members (Barchuk et al., 2004). These lig-ands include metabolites of JH-III, such as far-nesol, farnesoic acid, methyl farnesoate and juvenile hormone acid. Nonetheless, since the phylogenetic position of a receptor is clearly not correlated with its liganded/orphan status (Escriva et al., 2004), the confirmed existence of ligand-binding activity of Usp within the

in-sect members of ApLoTe group awaits experi-mental evidence.

4.2. High ecdysteroid titers seem to repress usp expression in stingless honeybees

To learn more of the existence of an occa-sional hormonal modulation of usp gene ex-pression and the relative participation of Usp protein in the vitellogenin gene expression reg-ulation and brain development in M. scutel-laris and S. depilis, we determined the expres-sion profile of usp in fat bodies (FB, where vg synthesis takes place) and brains during pu-pal development. The obtained profiles turned out not to be very similar between the species. In M. scutellaris FB and brain and in S. de-pilis brain usp expression anti-parallels pupal ecdysteroid titers, thus suggesting a repression of usp expression by this hormone. This is the case, for example, for Choristoneura fumifer-ana, where Cfusp seems to be down-regulated by high titers of 20E (Perera et al., 1998). Moreover, vg gene expression during pupal stages seems to be repressed by ecdysteroids in these bees, as has been shown in A. mellif-era (Barchuk et al., 2002), whose ecdysteroid titers during this period are similar to those of M. quadrifasciata and S. depilis (Hartfelder and Engels, 1998; Hartfelder et al., 2006). The observed profiles of ecdysteroids and usp ex-pression suggest a possible role of Usp pro-tein in vg expression regulation in these bees, at least in M. scutellaris.

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be an ecdysone receptor complex constituent, and so we would expect that its gene expres-sion would be induced by these hormones (see Auzoux-Bordenave et al., 2005; Tata, 2006). These results suggest that the usp gene is re-pressed by the mediating ecdysone receptor complex (EcR/Usp+20E) or that there is more than one isoform of Usp in these bees, where one of them is repressed by ecdysteroids and the other induced. This is true for Manduca sexta, where two Usp isoforms were found, Usp-1 and Usp-2, the first repressed by ecdys-teroids and the second induced (Hiruma et al., 1999). In bees, it has been demonstrated that A. mellifera possesses two usp transcripts dif-ferentially expressed in the bee’s body, one with∼ 4 kb and the other with ∼ 5 kb, and that usp gene expression is induced by JH, while ecdysteroids do not seem to have any ef-fect on the expression of this gene (Barchuk et al., 2004). In contrast, apoptosis processes during pupal brain development seem to be triggered by high ecdysteroid titers around the first stages of this developmental period (Ganeshina et al., 2000; Malun et al., 2003). The observed absence of the correspondent usp transcript during this period in M. scutel-laris and S. depilis (see Fig. 3; similar results were obtained in A. mellifera, ARB and ZLPS, unpubl. data) would mean its negative partic-ipation in this process or, again, its participa-tion through an unknown isoform.

In S. depilis, usp expression levels in brains are high during the first two pupal stages and fall abruptly in the third stage, thus differ-ing from the profile found in M. scutellaris (Fig. 3). Comparative morphological stud-ies of brain development including estimates of relative consequences of programmed cell death in both bee species are necessary to test possible differential brain development. The confirmation of this idea would have physio-logical implications, explaining for example, some aspects of the known differences in cell provisioning and feeding behaviors between Melipona and Scaptotrigona (see Michener, 1974). Additional and more accurate analyses of gene expression levels might help in deter-mining if these differences are meaningful.

The high levels of usp expression we found in the last pupal stage brains seem to represent

a response to an increase of JH titers which might occur during this period (see Hartfelder et al., 2006), as seen in A. mellifera (ARB and ZLPS, unpubl. data; Hartfelder and Engels, 1998). In this bee species, usp gene expres-sion is rapid and transiently up-regulated by JH, more likely after the formation of an ac-tive Usp-JH receptor complex (Barchuk et al., 2004, 2007; Guidugli et al., 2005a, b). Thus, the relationship between hormones and their nuclear receptors with the vg gene expres-sion regulation and brain development in bees (Dallacqua et al., 2006; Hartfelder et al., 2006; Barchuk et al., 2007) are challenging pro-cesses that need further investigation.

ACKNOWLEDGEMENTS

We would like to thank Sidnei Mateus and Dirk Louis Schorkopf for kindly providing stingless bees and João José dos Santos for technical assistance in the apiary of Ribeirão Preto. We also thank Adriana Mendes for technical assistance in LBDA. TRPM and ACAST were undergraduate re-cipients of research grants from Fapesp (Proc. No. 03/12073-0 and 03/12072-4). Financial support from Projeto Temático Fapesp (Proc. No. 2005/03926-5) is gratefully acknowledged.

Le gène ultraspiracle des abeilles sans aiguillon

Melipona scutellaris et Scaptotrigona depilis :

séquence de l’ADNc et profils d’expression au cours du développement nymphal.

ultraspiracle/ hormone juvénile / vitellogénine /

corps gras/ cerveau / abeille sans aiguillon

Zusammenfassung – Das Ultraspiracle Gen der Stachellosen Bienen Melipona scutellaris und Scaptotrigona depilis: cDNA Sequenz und Expressionsmuster während der Puppenpha-se. Die Entwicklung und Reproduktion von

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Expression von Zielgenen. Usp hat die Möglich-keit sowohl als Heterodimer eine Bindung an EcR oder HR38 (einem anderen nukleären Rezeptorpro-tein) einzugehen, als auch ein Homodimer mit ei-nem anderen Usp-Molekül zu bilden und dann JH zu binden. Damit kann Usp zu einem pleiotropen Faktor werden, der die Insektenentwicklung steuert, indem es die Wirkung beider Insektenhormone, der Ecdysteroide und von JH, in entscheidender Weise beeinflusst.

Die vorliegende Arbeit präsentiert Daten zur Cha-rakterisierung der ultraspiracle Komplementär-DNA (cKomplementär-DNA) von zwei Mitgliedern der Familie Apidae, der Stachellosen Bienen Melipona

scutel-laris und Scaptotrigona depilis. Unser Ziel war

es, Informationen über die entsprechenden Amino-säurensequenzen zu erhalten, die dann in evoluti-ve Analysen Eingang finden können. Insbesonde-re im Hinblick auf molekulaInsbesonde-re Mechanismen der Steuerung der Produktion des Dotterproteins Vitel-logenin untersuchten wir ausserdem die Expressi-onsmuster des usp Gens im pupalen Fettkörper und in Gehirnen von Arbeiterinnen dieser beiden Arten. Puppenstadien dieser Stachellosen Bienen wurden aus Kolonien entnommen, die im Meliponarium der Universidade de São Paulo, Ribeirão Preto, Brasili-en, gehalten wurden. Die Klonierung und Sequen-zierung der usp cDNAs erfolgte durch PCR Am-plifikation mittels spezifischer Primer für die Apis

mellifera usp cDNA Sequenz. Für die Bestimmung

der usp Expressionsmuster benutzten wir einen se-miquantitativen RT-PCR Ansatz.

Die usp cDNA Sequenzen für M. scutellaris und S.

depilis umfassen 1284, bzw. 1665 Basenpaare, die

für sehr ähnliche Polypeptide von 427 Aminosäu-ren kodieAminosäu-ren. Diese Werte und Sequenzen sind iden-tisch mit denen der Honigbiene und sehr ähnlich mit denen der Stechmücke Aedes aegypti, der Flie-ge Lucilia cuprina, des Reis-Stammbohrers Chilo

suppressalis und auch mit der grossen Usp-Isoform

der Schabe Blattella germanica.

Die usp Expressionsmuster zeigten erstaunlich we-nig Gemeinsamkeiten zwischen den beiden Bienen-arten, abgesehen von der Tatsache, dass die Trans-kriptionsraten im Gehirn und Fettkörper jeweils zu Beginn der Pupalentwicklung (Pw; siehe Tab. I und Abb. 3) am höchsten waren und zum Ende der Pup-penphase abnahmen. Interessanterweise zeigten die Expressionsmuster im Fettkörper und in Gehirnen von M. scutellaris und in Gehirnen von S. depilis einen entgegengesetzten zu den Ecdysteroid-Titern der Hämolymphe dieser beiden Arten, was auf ei-ne mögliche ei-negative Kontrolle der usp Expression durch diese Hormonklasse hinweist.

Bei S. depilis fanden wir in Gehirnen der frühen Pupenstadien eine hohe usp Transkriptionsrate, die dann in dem mittleren Stadium stark abfiel und da-mit stark von den bei M. scutellaris erda-mittelten Wer-ten abwich. (Abb. 3). Die hohen Werte der usp Expression in Gehirnen der späten Puppenstadien weisen auf eine Steuerung durch JH hin

(Hartfel-der et al., 2006), dessen Titer bei (Hartfel-der Honigbiene A.

mellifera in diesen Stadien langsam ansteigt.

ultraspiracle / Juvenilhormon / Ecdyson /

Vitellogenin/ Fettkörper / Gehirn

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